A. SPECIFIC AIMS Our efforts to determine how the Ssu72 CTD phosphatase component of the CPF complex interacts with the transcription initiation machinery led to our identification of "gene loops" that juxtapose promoter and terminator regions (Ansari and Hampsey, 2005;Singh and Hampsey, 2007). We detected loops at genes ranging in length from 1 kb to 7 kb, suggesting that looping might be a general phenomenon of RNAP II transcription. We propose that gene loops are dynamic chromatin structures that form in a transcription- dependent manner. Our working model is that gene loops promote transcription activation, perhaps by facilitating translocation of RNAP II from the terminator back to the promoter of the same gene, although we do not exclude other possibilities for how loops affect transcription. Herein, we propose to continue our studies of gene loops in yeast, focusing on the following three Specific Aims: Specific Aim #1: Define the physical characteristics of gene loops. To date, we have analyzed six genes for juxtaposition of promoter-terminator regions;all were chosen for analysis because of physical characteristics that made them amenable to study by 3C. The objectives of this Aim are to refine our physical analysis of looping and to identify genes that will facilitate physiological analysis of looping. It is not our intention to perform a global analysis of looping, but instead to identify loops at genes whose regulatory properties are well defined. The successful outcome of this Aim will provide us with target genes for assessing the structural requirements for looping under repressing and activating conditions (Aim #2) and the physiological consequences of looping (Aim #3). Specific Aim #2: Define the molecular determinants of gene looping. We will expand our analysis of factors that affect promoter-terminator interactions, asking if known components of the initiation and 3'-end processing machineries mediate looping. Our initial studies revealed that the transcription initiation factor TFIIB and the Ssu72 and Pta1 components of the CPF 3'-end processing complex are required for looping. In this Aim we will focus on (i) Sub1, a protein that affects initiation, but has also been reported to function as a RNAP II anti-terminator;(ii) the Gal4 and Pho4 transcriptional activators;and (iii) Rat1, a 5'->3'exonuclease involved in transcription termination. We will also screen additional initiation, 3'-end processing and termination factors for affects on looping, and propose to use our expertise in classical yeast genetics to search for other factors that affect promoter- terminator interactions. Specific Aim #3: Define the physiological significance of gene loops. We recently discovered that gene loops at GAL10 persist following a cycle of galactose activation and glucose repression and that persistence of looping is required rapid reactivation of GAL10 transcription. In Aim #3, we propose to confirm and extend these results. In addition to analysis of GAL10, we will determine whether looping affects activation/reactivation of the PHO5 gene. Our hypothesis is that looping is required for high levels of gene transcription and that looping underlies "memory" of recent transcriptional activity. The refinement of 3C technology offers a unique opportunity to determine how DNA topology regulates gene expression. The successful outcome of the experiments described in this proposal will provide novel insight into transcriptional regulatory mechanisms. Moreover conclusion from our studies with yeast can be extrapolated to better understand mammalian gene expression as several recent studies have identified comparable gene loops in human cells, including gene loops at the BRCA1 tumor suppressor gene and between the 5'LTR promoter and 3'LTR poly(A) signal of HIV, the causative agent of AIDS. PUBLIC HEALTH RELEVANCE: Several recent studies have revealed that DNA forms loops that juxtapose the ends of the gene. As examples, the human BRCA1 tumor suppressor gene and the HIV virus that causes AIDS were reported to form gene loops during their expression. We recently discovered that genes in yeast form similar DNA loops. We propose to use a powerful combination of molecular, genetic and biochemical methods to study gene loops in yeast to provide insight into gene expression mechanisms that go awry in cancers and other human diseases.